One of the persistent problems involved in the recombinant production of desired proteins resides in the obtainment of proper processing and folding of the recombinant product. Because the gene encoding the primary protein structure is generally expressed in a host system foreign to that in which it is normally transcribed and translated, the resultant product protein, while having the correct amino acid sequence, has different characteristics from the native protein. In some instances, a part of this difference resides in a change in molecular structure--most commonly, lack of normally associated glycosylation. However, it is also understood that the three-dimensional conformation of the protein may be different depending on the cellular environment. In particular, various recombinant hosts are thought to have differing environments with respect to the level of oxidation or reduction that normally is associated with the cellular components, resulting in recombinant proteins with disulfide bond compositions which differ from those of the native protein, and, in general, altered three-dimensional dimensional structures. These alterations may have deleterious effects on behavior and activity.
In the case of basic fibroblast growth factor (bFGF), glycosylation patterns are not germane as the native molecule is unglycosylated. However, recombinantly produced material differs from that isolated from pituitaries in its chromatographic behavior and poses stability problems unless measures are taken to prevent multimerization in solution. It has been shown, by applicant herein, and as disclosed by Seno, M., et al., Biochem Biophys Res Comm (1988) 151:701, that substitution of serine for the cysteines at positions 78 and 96 prevents multimerization. It is also the case that bFGF isolated from natural bovine pituitary does not multimerize.
It has been disclosed that amino acid composition determinations of native isolated bovine bFGF indicate the presence of six cysteines, although the gene encodes only four. It was proposed that additional residues of cysteine might be disulfide linked to the protein (Esch, F., et al., Proc Natl Acad Sci USA (1985) 82:6507).
The formation of conjugates of cysteine or glutathione with proteins by reaction with cystine or glutathione disulfide as a natural phenomenon has long been suggested and disclosed. In 1960, Eagle, H. et al., J Biol Chem (1960) 235:1719-1726 showed that mammalian cell cultures were abetted by addition of compounds capable of forming S--S covalent bonds including, e.g., cysteine, S.sub.2 O.sub.3.sup.-2, and thioglycolate. The authors concluded that the proteins in the medium were bound by S--S bonds to these reagents. Human serum albumin was disclosed to exist in a dimerized form, presumably disulfide bonded, and in a monomeric form stabilized by cysteine or glutathione by King, T. P., J Biol Chem (1961) 236:PC5. Binding to glutathione was proposed to account for the heterogeneity of hemoglobin by Huisman, T. H. et al., J Lab & Clin Med (1962) 60:302-319.
Hanap, K. R. et al., Biochim Biophys Acta (1973) . 310:104-110 acknowledged the presence of mixed disulfides of glutathione and serum albumin and found them reducible by glutathione reductase. Isaacs, J. et al., Biochim Biophys Acta (1977) 497:192-204 propose formation of mixed disulfides of proteins with glutathione as a means to regulate S--S/SH ratio. Conversely, Mannervitc, B. et al., Biochim J (1980) 190:125-130 postulate the formation of disulfides with proteins to represent a mechanism for regulation of protein activity. Pyruvate kinase is used as an example.
It has also been proposed, because of the conservation of cysteines at residues 34 and 101 in the genes encoding bFGF in various mammals, that an intramolecular disulfide is formed between these two residues, and the formation of this disulfide in the recombinant mutant form of bFGF having serine substituted for cysteine at positions 78 and 96 has been reported (Fox, G. M., et al., J Biol Chem (1988) 263:18452).
It has now been found that addition of an organic disulfide-containing compound, such as glutathione disulfide, to preparations of recombinantly produced bFGF results in enhanced stabilization and behavior more closely related to the native protein. Purification of this stabilized form of bFGF results in a preparation suitable for formulation for pharmaceutical applications.